PerspectiveCANCER

Reprogramming to resist

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Science  06 Jan 2017:
Vol. 355, Issue 6320, pp. 29-30
DOI: 10.1126/science.aam5355

One means by which cancer cells evade therapies involves their ability to reprogram to a cell type that no longer depends on the cellular pathway being targeted by the treatments. Hormone deprivation therapies that suppress androgen receptor (AR) signaling are the mainstay of treatment for metastatic prostate cancer. However, prostate cancers can become resistant to this approach by losing dependence on androgen hormones. On pages 84 and 78 of this issue, Mu et al. (1) and Ku et al. (2), respectively, contribute to our mechanistic understanding of this remarkable plasticity in cell identity, which allows cancers to thrive.

Androgens stimulate prostate cancer cell growth. The main androgens are testosterone and dihydrotestosterone, which are synthesized primarily in the testes. Decreasing androgen production or preventing the hormones from acting on prostate cancer cells often makes the tumors shrink or grow more slowly. However, prostate cancer can adapt to androgen deprivation through alterations that restore AR signaling and maintain their luminal epithelial adenocarcinoma phenotype, even when androgen production is low [referred to as castration-resistant prostate cancer-adeno (CRPC-adeno)] (3). With the development of more effective AR-targeting drugs such as abiraterone and enzalutamide, additional resistance mechanisms are arising. About a quarter of these resistant tumors undergo cellular reprogramming and acquire a continuum of neuroendocrine characteristics (CRPC-NE) (4, 5). Genomic analyses have shown that CRPC-NE evolves from CRPC-adeno. Most CRPC-NE express one or more NE-lineage markers [such as synaptophysin (SYP)], and there are a range of morphological variants, perhaps reflecting variable differentiation states. The increased expression of AR and AR-regulated genes is generally reduced in CRPC-NE compared to CRPC-adeno, although there is a range of overlap that may reflect ongoing selection as well as differences in genomics (6).

In addition to NE-lineage markers, the messenger RNA profiles (transcriptomes) of CRPC-NE patient samples and prostate cancer models have shown increased expression of genes involved in neuronal development, such as sex determining region Y box 2 (SOX2), and genes encoding epigenetic regulators, such as enhancer of zeste homology 2 (EZH2) and DNA methyl-transferase 1 (DNMT1) (6, 7). DNMT1 may contribute to epigenetic characteristics, such as DNA methylation patterns, that are markedly different between CRPC-adeno and CRPC-NE (6). In the most comprehensive genomic analysis of CRPC-NE to date (6), the co-occurrence of alterations in cell signaling pathways involving the tumor suppressor proteins retinoblastoma 1 (RB1) and tumor protein 53 (TP53) was highly enriched in CRPC-NE (∼50%) relative to CRPC-adeno (ů15%), suggesting the involvement of these pathways in the selection of CRPC-NE. Mu et al. and Ku et al. connect the loss of the RB1 and TP53 genes to lineage plasticity and epigenetic regulation in prostate cancer resistance to androgen deprivation therapy.

Mu et al. addressed the phenotypic consequences of RB1 and TP53 silencing in a human cell line that overexpresses AR (LNCaP-AR cells), a model of CRPC-adeno that is sensitive to the AR antagonist enzalutamide. Silencing both RB1 and TP53, but neither alone, caused marked enzalutamide resistance in these cells, although AR activity persisted and remained responsive to enzalutamide. Notably, cells lacking TP53 and RB1 displayed lineage plasticity, as indicated by decreased expression of luminal epithelial cell markers and increased expression of basal epithelial cell and neuroendocrine markers. Moreover, these gene expression changes occurred within 48 hours of induced depletion of TP53 and RB1 and could be rapidly reversed, indicating a direct effect rather than selection for cells with preexisting phenotypic differences. Mu et al. used RNA-sequencing data sets of clinical samples to identify transcription factors whose expression correlated with co-occurring alterations in the RB1 and TP53 signaling pathways. Among these, SOX2 expression was induced by silencing RB1 and TP53 in LNCaP-AR cells, and SOX2 expression in these cells was necessary and sufficient for the expression of basal epithelial and neuroendocrine markers as well as for enzalutamide resistance. RB1 loss has been associated with lineage plasticity in other cancer cell models, and in induced pluripotent stem cells, RB1 inhibited the basal transcription of SOX2 and other pluripotency genes (8). These data suggest a model whereby the induction of SOX2 expression subsequent to RB1 and TP53 loss contributes to neuroendocrine differentiation and AR-pathway independence.

A model of progressive reprogrammingGRAPHIC: K. SUTLIFF/SCIENCE

Ku et al. examined the effects of Rb1 and Tp53 deletion in a mouse model of metastatic prostate cancer that is based on the lack of phosphatase and tensin homolog (Pten), another tumor suppressor. Prostatespecific deletion of Pten led to adenocarcinoma (expressing AR) as expected, whereas the simultaneous deletion of Rb1 produced an aggressive metastatic disease that was initially an adenocarcinoma but progressed to heterogeneous histology with markers of adenocarcinoma and neuroendocrine disease, including variable AR expression. Most tumors responded transiently to castration (a reduction in androgen production), and two of five recurrent tumors that were analyzed contained Tp53 mutations, suggesting a role for Tp53 loss in castrate resistance. Gene expression analysis showed marked overlap between the differentially expressed genes in mice lacking Pten and Rb1 and mice lacking Pten, Rb1, and Tp53. This overlapping set of genes was similar to the genes observed in human neuroendocrine prostate cancer, indicating that Rb1 loss was the major driver of this apparent differentiation. These changes included increased expression of genes regulated by the transcription factor E2F (including Sox2 and Ezh2), neuroendocrine lineage genes, and genes related to stem cells and epigenetic reprogramming. Ezh2 inhibition or silencing increased AR and decreased Syp expression in cell lines derived from mouse prostate tumors lacking Pten and Rb1. These cell lines also became sensitive to inhibition by enzalutamide. Similarly, Ezh2 inhibition restored enzalutamide responsiveness in the RB1- and TP53-depleted LNCaP-AR cells in the study by Mu et al.

The studies by Mu et al. and Ku et al. indicate that RB1 loss not only facilitates the outgrowth of neuroendocrine-like cells that have decreased AR signaling but also facilitates lineage reprogramming by stimulating expression of SOX2. The studies also suggest a role for TP53 loss, although RB1 loss alone appears sufficient to drive neuroendocrine differentiation in the mouse model, perhaps related to the presence of less-differentiated luminal epithelial cells. Further studies in additional metastatic prostate cancer models will be important to assess the generalizability of these results. Furthermore, RB1 loss may contribute other functional effects. RB1 loss alone can confer resistance to AR-targeted therapies (9). The extent to which TP53 or RB1 loss directly contributes to regulating AR expression and/or activity observed in neuroendocrine prostate cancer is also unclear. Indeed, RB1 loss may directly enhance AR expression and signaling (10). Nonetheless, results in the mouse model indicate that Rb1 loss does not directly suppress AR and instead suggest that there may be a selective advantage to decreasing AR expression once cell growth becomes AR independent.

SOX2, an E2F-regulated gene, was identified as a downstream mediator of RB1-dependent lineage plasticity. SOX2 plays context-dependent roles in both maintaining pluripotency and driving neuronal progenitor differentiation (11). It will be important to determine the various roles for SOX2 and to what extent in vitro modeling can be generalized to disease progression in vivo. Moreover, the effects of EZH2 inhibition are a key finding and parallel other studies showing the importance of EZH2-dependent mechanisms in supporting CRPC-NE growth, including models initiated by other drivers (12). Notably, EZH2 is also a direct AR coactivator (13). This suggests that EZH2 inhibition in combination with other therapeutics, including enzalutamide, has the potential to increase responses in patients with CRPC-NE disease.

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